Folate, as part of the one carbon cycle, is critical for the de novo synthesis of S-adenosyl methionine (SAM). SAM, in turn, provides the methyl group for DNA methylation, a key mode of epigenetic regulation. Studies have shown that manipulation of folate levels during gestation alters the epigenetic status of genes, and that treatment with methyl donors in adulthood reverses DNA methylation changes. Given the effect of folate levels on DNA methylation, it is not surprising that dietary folate status influences the risk for several cancers. Folate is also essential for cell proliferation. Accordingly, animal studies show that the timing of folate intake modulates disease outcome: supplementation protects against tumor initiation while folate depletion inhibits tumor growth. The prostate has a high requirement for folate, and also seems susceptible to alterations in DNA methylation, suggesting that folate might also play a role in prostate cancer. We have developed an in vitro model, in which we can produce simultaneous tumor suppressor gene promoter hypermethylation in an overall context of global hypomethylation reminiscent of prostate cancer, by manipulating folic acid levels. In addition, we have shown that a protein intimately related to intracellular folate levels, Prostate-Specific Membrane Antigen (PSMA), contributes to prostate carcinogenesis. PSMA, a unique folate hydrolase, and possible folate transporter, undergoes significant up-regulation in prostate cancer and in the endothelial cells of tumor neovasculature. Moreover, in the presence of low levels of folate, PSMA expression increases cell invasiveness, an activity important for both tumor formation and progression. Our hypothesis is: Low, followed by excess levels of available folates in the prostate over an extended period of time leads to carcinogenesis. To test this hypothesis we will use the following specific aims; 1) Determine if (a) PSMA expression regulates intracellular folate levels, and (b) if folate levels regulate DNA methylation in prostate tissues in a novel in vivo model, 2) Ascertain if low levels of folates increase the invasive capacity of PSMA, and lead to genomic hypomethylation, DNA instability and subsequently carcinogenesis and 3) Establish if, prior to initiation of prostate carcinogenesis folate supplementation is protective, and if, following initiation, folate supplementation promotes prostate tumor growth and progression by enhanced uptake of systemic folate in the prostate mediated by PSMA, and by altering epigenetic programming. Relevance: Recent reports describe a significant association between folate supplementation and prostate cancer. Folic acid fortification of the U.S.diet in 1998 has converted the population from a largely folate-deficient to folate-replete. Studies have shown that excess folic acid intake increases the risk of breast, colorectal, and now, prostate cancer. We have shown that nearly 2 million men aged 60 or above have serum folate levels greater than 5 fold adequate. This fact, in combination with the high prevalence of preclinical prostate cancer in men of the >60 age group, underscores the importance of investigating the relationship between folate and prostate cancer. PUBLIC HEALTH RELEVANCE: These studies have the potential to yield important information regarding both the safety of, and the potentially protective effects, of folic acid supplementation on prostate carcinogenesis and progression, and are particularly timely given the recent mandatory fortification of the American diet with folic acid. Dietary folate intake is an easily modifiable factor; prostate cancer is a slow growing disease - even if we can effect only a modest increase in time to progression via dietary folate intake, it could result in a large clinical impact. If we understand how dietary folate manipulation can alter DNA methylation patterns, and how the effect of folate may be modulated by PSMA expression, we may be able to detect and provide nutritional or chemo- intervention to men at-risk for prostate cancer, preventing or delaying the disease.